Analysis of candidate genes through a proteomics-based approach in primary cell lines from malignant melanomas and their metastases

Franco Carta, Pietro R. Demuro, Cristina Zanini, Antonella Santona, Daniele Castiglia, Stefania D'Atri, Paolo A. Ascierto, Maria Napolitano, Antonio Cossu, Bruna Tadolini, Franco Turrini, Antonella Manca, Maria C. Sini, Giuseppe Palmieri, Carla Rozzo

Research output: Contribution to journalArticlepeer-review

Abstract

Proteomics provides a powerful approach for screening alterations in protein expression and post-translational modification associated with particular human diseases. In this study, the analysis of protein expression was focused on malignant melanoma in order to determine the candidate genes involved in tumour progression. The proteomes of cultured melanocytes and of cell lines from primary and metastatic lesions of one malignant melanoma patient were profiled using two-dimensional electrophoresis (2-DE) and mass spectrometry. Differentially expressed proteins were confirmed by 2-DE and mass spectrometry on an additional four malignant melanoma cell lines. Total RNA from the first subset of cell lines was used for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the candidate genes identified after proteomics analysis. A very high similarity was observed in the 2-DE maps of two malignant melanoma cell lines derived from primary and secondary lesions of the same patient. Mass spectrometry identified 37 proteins which were found to be more abundant in tumour cells in comparison with control melanocytes (as confirmed on additional cell lines), with a relatively high prevalence of stress proteins. Eight candidate genes (PRDX2, HSP27, HSP60, HSPA8, HSP9B, STIP1, PDI and P4HB) were further characterized by evaluating their messenger RNA expression levels through real-time RT-PCR analysis. Overexpression of HSP27, HSP60 and HSPA8 and downregulation of PRDX2 were observed in cells from metastatic malignant melanoma in comparison with those from primary melanoma. Although further investigations with larger numbers of paired normal and tumour samples are needed, our findings strongly suggest that the dysregulation of stress pathways may be involved in melanoma progression.

Original languageEnglish
Pages (from-to)235-244
Number of pages10
JournalMelanoma Research
Volume15
Issue number4
DOIs
Publication statusPublished - Aug 2005

Keywords

  • Expression analysis
  • Malignant melanoma
  • Proteomics
  • Quantitative reverse transcriptase-polymerase chain reaction
  • Tumour progression

ASJC Scopus subject areas

  • Cancer Research
  • Dermatology

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